Calcium regulates seed germination under saline stress

Researchers at the Hebrew University of Jerusalem have identified a molecular mechanism involved in controlling the balance between sodium and potassium during germination under saline stress. The system involves calcium signaling and the coordinated action of the proteins CAMTA6, PP2C49, and HKT1;1. Experiments with Arabidopsis thaliana also showed increased germination after application of sanguinarine, an inhibitor of PP2C phosphatases. However, the effect was limited to germination and did not protect established seedlings against salinity.

The study evaluated germinating Arabidopsis thaliana embryos (doi 10.1111/tpj.71033). The researchers observed a spatial organization of the response to salt. CAMTA6 expression predominated in the cotyledons. Under salinity, gene activity increased and concentrated at the cotyledon margins. HKT1;1 expression showed a different pattern. In the presence of both sodium chloride and calcium chloride, HKT1;1 concentrated in the radicle.

Calcium and calmodulin signaling

CAMTA6 functions as a transcription factor associated with calcium and calmodulin signaling. The results indicate the participation of this protein in the spatial control of HKT1;1. The HKT1;1 transporter participates in sodium and potassium homeostasis. The distribution of its expression in the embryo influences the seed's ability to maintain ionic concentrations compatible with germination under salinity.

The phosphatase PP2C49 integrates the same system. Previous studies already indicated its ability to inactivate HKT1;1 by dephosphorylation. In the new work, the activity of the PP2C49 promoter remained concentrated in the radicle of wild-type plants. In mutants without functional CAMTA6, expression spread to other tissues of the embryo. The result points to the participation of CAMTA6 in the spatial restriction of PP2C49 expression.

Sanguinarine effect

The researchers also evaluated the effect of sanguinarine, a benzophenanthridine alkaloid with inhibitory activity on PP2Cs. In wild-type seeds subjected to 150 millimoles per liter of sodium chloride, the application of one micromole per liter of sanguinarine increased germination from 76% to 92%. Under 200 millimoles per liter of sodium chloride, the rate increased from 3% to 37%.

The response depended on the CAMTA6-PP2C49-HKT1;1 axis. The improvement caused by sanguinarine was compromised in mutants of camta6, pp2c49, and hkt1, as well as combinations of these mutations. The loss of HKT1;1 eliminated the favorable response to the compound in some tested lines. According to the researchers, the data support the transporter's role in sanguinarine's effect on tolerance during germination.

System components

The application of the compound also modified the expression of the system's components. In wild-type embryos, sanguinarine reduced the expression of CAMTA6 and PP2C49. At the same time, it increased the expression of HKT1;1. Under salinity, this alteration coincided with an improvement in the ionic composition of the embryos.

With 150 millimoles per liter of sodium chloride, wild-type embryos accumulated 2,61 micrograms of sodium per embryo. The combination of salt with sanguinarine reduced this value to 1,89 micrograms. For potassium, the content increased from 0,85 micrograms to 1,10 micrograms per embryo. The treatment, therefore, increased the potassium-to-sodium ratio during germination.

The effect did not appear in hkt1 mutants. Under sodium chloride, these embryos accumulated 3,10 micrograms of sodium per embryo. With sanguinarine, the value remained close, at 3,06 micrograms. The potassium concentration also remained low. These results reinforce the need for HKT1;1 for the response observed in the wild type.

Time limit

The action of sanguinarine showed a time limit. Seedlings three days old, transferred to media containing salt and sanguinarine, did not maintain the benefit observed during germination. The compound did not prevent the reduction in primary root growth or the loss of chlorophyll caused by saline stress. The researchers conclude, therefore, based on the tests carried out, that there is a specific effect for the germination phase.

Transcriptome analysis broadened the scope of the mechanism. The group identified 63 PP2C phosphatase genes that responded to the evaluated conditions. Some of them showed regulation associated with CAMTA6, calcium, and sodium chloride. The data indicate a broader signaling network than the axis formed by CAMTA6, PP2C49, and HKT1;1.

The results do not yet demonstrate a technology ready for agricultural use. The study used Arabidopsis thaliana and focused its tests on the molecular functioning of germination. The team points to this mechanism as a basis for future research on genes and strategies capable of favoring crop establishment in saline environments.